title: Establishing catalytic activity on an artificial (βα)8-barrel protein designed from identical half-barrels.
abstract: It has been postulated that the ubiquitous (βα)8-barrel enzyme fold has evolved by duplication and fusion of an ancestral (βα)4-half-barrel. We have previously reconstructed this process in the laboratory by fusing two copies of the C-terminal half-barrel HisF-C of imidazole glycerol phosphate synthase (HisF). The resulting construct HisF-CC was stepwise stabilized to Sym1 and Sym2, which are extremely robust but catalytically inert proteins. Here, we report on the generation of a circular permutant of Sym2 and the establishment of a sugar isomerization reaction on its scaffold. Our results demonstrate that duplication and mutagenesis of (βα)4-half-barrels can readily lead to a stable and catalytically active (βα)8-barrel enzyme.
authors.affiliation: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Josef M
authors.initials: JM
authors.lastname: Sperl
authors.forename: Bettina
authors.initials: B
authors.lastname: Rohweder
authors.forename: Chitra
authors.initials: C
authors.lastname: Rajendran
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000619:Aminohydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases
keywords: (βα)(8)-Barrel; Artificial enzyme; Enzyme design; Enzyme evolution